Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Fatal leptospirosis in endangered Barbary macaques (Macaca sylvanus) kept in captivity: Assessing the role of sympatric rodents.

Between December 2020 and January 2021, an outbreak of acute mortality in endangered Barbary macaques (Macaca sylvanus) kept in captivity was detected in a zoo in Spain. The main findings observed in the two fatally affected animals at post-mortem evaluation were jaundice, renal tubular necrosis and interstitial nephritis. Leptospira spp. infection was confirmed by real time PCR (qPCR) in different tissues in both individuals. Analyses of secY gene from a positive individual showed 100% homology with a previously published sequence corresponding to Leptospira interrogans serovar Copenhageni. Free-living sympatric brown rats (Rattus norvegicus) from the affected zoo were also analyzed, and showed a prevalence and seroprevalence of Leptospira spp. of 18.2% (4/22; 95% CI: 2.1-34.3) and 41.9% (26/62; 95% CI: 29.7-54.2), respectively. We detected seropositive sera to five different serovars of Leptospira spp. (Copenhageni, Grippotyphosa, Pomona, Canicola and Hardjo) in the rodent population, with L. Copenhageni being the predominant one. This study describes for first time an outbreak of fatal leptospirosis in captive non-human primates in Europe. Our results show that Barbary macaques, an endangered species, are highly susceptible to Leptospira spp. infection, with sympatric wild rodents being the most likely reservoir animals involved in transmission in this outbreak. Our results suggest that rodent control could be an effective measure for minimizing exposure to Leptospira spp. in zoological collections. Given the potential implications for conservation, animal and public health, non-human primates and rodents should be included in surveillance programs for Leptospira spp. in zoos.

Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks, Spain, October 2022.

In October 2022, an outbreak in Europe of highly pathogenic avian influenza (HPAI) A(H5N1) in intensively farmed minks occurred in northwest Spain. A single mink farm hosting more than 50,000 minks was involved. The identified viruses belong to clade 2.3.4.4b, which is responsible of the ongoing epizootic in Europe. An uncommon mutation (T271A) in the PB2 gene with potential public health implications was found. Our investigations indicate onward mink transmission of the virus may have occurred in the affected farm.

Clinical, Virological and Immunological Responses after Experimental Infection with African Horse Sickness Virus Serotype 9 in Immunologically Naïve and Vaccinated Horses.

This study described the clinical, virological, and serological responses of immunologically naïve and vaccinated horses to African horse sickness virus (AHSV) serotype 9. Naïve horses developed a clinical picture resembling the cardiac form of African horse sickness. This was characterized by inappetence, reduced activity, and hyperthermia leading to lethargy and immobility-recumbency by days 9-10 post-infection, an end-point criteria for euthanasia. After challenge, unvaccinated horses were viremic from days 3 or 4 post-infection till euthanasia, as detected by serogroup-specific (GS) real time RT-PCR (rRT-PCR) and virus isolation. Virus isolation, antigen ELISA, and GS-rRT-PCR also demonstrated high sensitivity in the post-mortem detection of the pathogen. After infection, serogroup-specific VP7 antibodies were undetectable by blocking ELISA (b-ELISA) in 2 out of 3 unvaccinated horses during the course of the disease (9-10 dpi). Vaccinated horses did not show significant side effects post-vaccination and were largely asymptomatic after the AHSV-9 challenge. VP7-specific antibodies could not be detected by the b-ELISA until day 21 and day 30 post-inoculation, respectively. Virus neutralizing antibody titres were low or even undetectable for specific serotypes in the vaccinated horses. Virus isolation and GS-rRT-PCR detected the presence of AHSV vaccine strains genomes and infectious vaccine virus after vaccination and challenge. This study established an experimental infection model of AHSV-9 in horses and characterized the main clinical, virological, and immunological parameters in both immunologically naïve and vaccinated horses using standardized bio-assays.

Seroprevalence of Leptospirosis, Brucellosis, and Q Fever in a Wild Red Deer (Cervus elaphus) Population Kept in a Fenced Reserve in Absence of Contact with Livestock.

Wildlife health is of interest for public and animal health because wild animals have been identified as important sentinels for the surveillance for zoonotic pathogens. This work investigated Brucella spp., Coxiella burnetii, and Leptospira spp. infection seroprevalence in a free-ranging red deer population. The study was conducted in a fenced reserve with controlled hunting activity in central Spain with animals that did not have any contact with livestock. Sampling was performed at two time points before and 5 years after the implementation of new management measures, including a reduction in the red deer population in the reserve. In addition, the presence of Leptospira DNA was tested in placental and fetal samples from seropositive pregnant animals. Antibodies against Brucella and Coxiella were not detected in any sample. The seroprevalence of Leptospira was 9.4% (13/137) in the first sampling for serovars Canicola and Panama. Five years later, the prevalence rose to 38.5% (97/252) with Pomona, the only serovar detected. Animals older than 2 years (50%; 70/140) were more likely to be Pomona seropositive than animals ≤2 years old (25.2%; 27/107; p < 0.001). Leptospira DNA was not detected in any sample tested. In conclusion, wild red deer in this area without contact with livestock seem not to play an important role in Brucella spp. and C. burnetii maintenance. The high seroprevalence of Leptospira spp. serogroup Pomona could indicate a risk for people with narrow contact with these animals, but the carrier status was not assessed. Consequently, it is unknown if red deer would represent a risk for human infection. Considering that wild boar could be the source of infection to red deer, the role of wild boar in the spread of leptospirosis and the risk for human infection should be investigated.

Epidemiological surveillance of bluetongue virus serotypes 1, 4 and 8 in Spanish ibex (Capra pyrenaica hispanica) in southern Spain.

A cross-sectional study was carried out to assess the prevalence and circulation of bluetongue virus (BTV) in Spanish ibexes (Capra pyrenaica hispanica). A total of 770 sera samples, 380 blood samples and 34 spleen samples were collected between 2006 and 2009 in Andalusia (southern Spain), a region and time period with a wide circulation of BTV in livestock. Thirty-one out of 770 (4.0%; CI(95%): 2.6-5.4) sera samples analyzed by ELISA showed antibodies against BTV. Twenty-four out of 31 seropositive samples were tested against BTV serotypes 1, 4 and 8 by serum neutralization test (SNT). Neutralizing antibodies against BTV-1 and BTV-4 were detected in seven and ten animals, respectively, four of them showed neutralizing antibodies to both serotypes. The animals seropositive to BTV-4 were sampled between 2006 and 2008, while BTV-1 circulation was confirmed in ibexes sampled between 2007 and 2009. None of the ibexes presented neutralizing antibodies against BTV-8. Statistically significant differences were found among regions and years, which is in coincidence with what occurred in domestic ruminants. There were no statistically significant differences between sexes, age classes and habitats (captivity vs. free-living). BTV RNA was not found in any of the 380 blood samples analyzed. However, BTV-1 RNA was detected from spleen in one Spanish ibex from Málaga province in August 2008. This finding evidences the presence of BTV-1 in Spanish ibex in a municipality where BT outbreaks were not detected in domestic ruminants during that period. Results of the present study show that Spanish ibexes were exposed and responded serologically to both BTV-1 and BTV-4. The low seroprevalence obtained suggests that Spanish ibex is not a relevant species in the dissemination of BT. However, the detection of BTV-1 RNA and the presence of seropositive ibexes in areas where BT outbreaks were not detected in livestock, could not exclude a significant role in the epidemiology of BTV in certain areas.

Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation.

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

High throughput detection of bluetongue virus by a new real-time fluorogenic reverse transcription-polymerase chain reaction: application on clinical samples from current Mediterranean outbreaks.

A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of bluetongue virus (BTV) in blood samples. A combination of primers specific for a highly conserved region in RNA segment 5 (based on Mediterranean BTV sequences) and a DNA probe bound to 5'-Taq nuclease-3' minor groove binder (TaqMan MGB) was used to detect a range of isolates. This real-time RT-PCR assay could detect 5.4 x 10(-3) tissue culture infectious doses (TCID50) of virus per milliliter of sample, which was comparable to our current BTV diagnostic nested RT-PCR assay. The assay detected all recent Mediterranean isolates (including serotypes 2, 4, and 16), BTV vaccine strains for serotypes 2 and 4, and 15 out of the 24 BTV reference strains available (all serotypes), but did not detect the related orbiviruses epizootic hemorrhagic disease and African horse sickness viruses. Following assay evaluation, the ability of this assay to identify BTV in recent isolates (2003, 2004) from ovine and bovine samples from an epizootic outbreak in Spain was also tested. Minor nucleotide changes (detected by sequencing viral genomes) within the probe-binding region were found to have a profound effect on virus detection. This assay has the benefits of being fast and simple, and the 96-well format enables large-scale epidemiological screening for BTV, especially when combined with a high-throughput nucleic acid extraction method.

Endogenous calmodulin interacts with the epidermal growth factor receptor in living cells.

We have previously shown that exogenous calmodulin (CaM) binds to the epidermal growth factor receptor (EGFR) at its cytosolic juxtamembrane region inhibiting its tyrosine kinase activity. We demonstrate in this report that endogenous CaM binds to EGFR in intact cells as CaM co-immunoprecipitates with EGF-activated and non-activated receptors. We also show in living cells that cell-permeable CaM inhibitors prevent the full transphosphorylation of wild type EGFR but not the transphosphorylation of an insertional EGFR mutant in which the CaM-binding domain was divided into two parts. Overall these results suggest that CaM interacts with EGFR in vivo.

Antiproliferative effect of nitric oxide on epidermal growth factor-responsive human neuroblastoma cells.

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.